STAT3 induces transcription of the DNA methyltransferase 1 gene (DNMT1) in malignant T lymphocytes

Abstract

In this study, we demonstrated that STAT3, a well-characterized transcription factor expressed in continuously activated oncogenic form in the large spectrum of cancer types, induces in malignant T lymphocytes the expression of DNMT1, the key effector of epigenetic gene silencing. STAT3 binds in vitro to 2 STAT3 SIE/GAS-binding sites identified in promoter 1 and enhancer 1 of the DNMT1 gene. STAT3 also binds to the promoter 1 region and induces its activity in vivo. Treatment of the malignant T lymphocytes with STAT3 siRNA abrogates expression of DNMT1, inhibits cell growth, and induces programmed cell death. In turn, inhibition of DNMT1 by a small molecule inhibitor, 5-aza-2-deoxy-cytidine, and 2 DNMT1 antisense DNA oligonucleotides inhibits the phosphorylation of STAT3. These data indicate that STAT3 may in part transform cells by fostering epigenetic silencing of tumor-suppressor genes. They also indicate that by inducing DNMT1, STAT3 facilitates its own persistent activation in malignant T cells. Finally, these data provide further rationale for therapeutically targeting STAT3 in T-cell lymphomas and, possibly, other malignancies.

Figure 1.

Coexpression of activated STAT3 and DNMT1 in malignant T-cell lymphoma cells. (A) Western blot analysis with the depicted antibodies of the nuclear protein extracts from cell lines derived from T-cell lymphomas involving skin (PB-1, 2A, 2B), HTLV-I–associated (HUT102B), and anaplastic lymphoma kinase–positive (SUDHL-1, JB6, Karpas 299) malignancies. Normal T-cell–rich PBMCs, both resting and PHA activated, and B cells (RAJI and DAUDI) served as controls. (B) Immunohistochemical analysis of the cutaneous T-cell lymphoma tissues. (left) Hematoxylin-eosin staining was used to visualize malignant T cells. (middle) Anti–phospho(p)-Y705STAT3 staining. (right) Anti-DNMT1 staining. Original magnification: larger images, 100 ×; insets, 400 ×. Depicted results that show coexpression of p-STAT3 and DNMT1 by malignant T cells are representative for all biopsy tissue samples of the 15 examined patients. (C) RT-PCR analysis of DNMT1 mRNA expression in the same diverse T-cell lymphoma cell line depicted in Figure 1A, with PBMCs serving as control.

Figure 2.

Association of STAT3 with SIE/GAS sites identified in DNMT1 gene promoter 1 and enhancer 1 in vitro. (A) Map of the DNMT1 gene promoter and enhancer region with highlighted STAT3-binding sites and corresponding DNA oligonucleotide probes used in EMSA and primer sets used in ChIP (for comparison, see Figure 4). (B) EMSA with DNA oligonucleotides that corresponded to promoter 1 (P1) and enhancer 1 (E1) STAT3-binding sites with nuclear protein extracts from malignant T cells 2A and normal PBMCs as control. (C) Binding of the 2A T-cell line protein extracts (protein) to the enhancer 1 and promoter 1 STAT3-binding sites in the presence and absence of the unlabeled (cold and SHP-1) probes. (D) Binding of the 2A and 2B T-cell line protein extracts to promoter 1 and enhancer 1 STAT-binding sites either wild-type (P1 and E1, respectively) or with mutated (M) TT and AA pairs by substitution with the CC pairs. (E) Supershift EMSA with the anti-STAT3 and control anti–anaplastic lymphoma kinase (ALK) antibody.

Figure 3.

Binding and activation of STAT3 to DNMT1 gene promoter/enhancer region in vivo. Cell lysates from T-cell lymphoma cell lines 2A and 2B were examined in the chromatin immunoprecipitation (ChIP) assay before (input) and after immunoprecipitation using anti-STAT3 antibody with an unrelated (ALK) antibody serving as the negative control. DNA binding was detected in the ChIP assay using PCR primer pairs corresponding to promoter 1 (A, P1) and enhancer 1 (B, E1) regions. Binding to the 3′-end of the DNMT1 gene served as an additional negative control. (C) STAT3 effect on activity of the DNMT1 promoter and enhancer. SUDHL-1 cells that constitutively expressed activated STAT3 were transfected with firefly luciferase constructs containing promoter 1 and enhancer 1 of the DNMT1 gene in either wild-type form (P1 and E1, respectively) or with mutated (M) TT and AA pairs that were substituted with the CC pairs. Results are presented in relative luciferase unit (RLU) after normalization based on expression of the cotransfected renilla luciferase gene.

Figure 4.

STAT3 depletion by siRNA inhibits expression of DNMT1. (A) Expression of STAT3, DNMT1, and the other listed proteins after treatment of the T-cell lymphoma cell lines 2A and 2B for the indicated times with STAT3 siRNA or control, non–sense (NS) siRNA. Untreated PB-1 line served as an additional control. (B) STAT3 siRNA–mediated inhibition of cell growth of 2A cells as determined in the MTT enzymatic conversion assay. Time point (TP) 1 designates a single siRNA treatment at the experiment inception (0 hour), and TP2 designates 2 treatments at 0 hour and at 48 hours. The assay readout was performed at 72 hours. (C) STAT3 siRNA–mediated induction of apoptotic cell death as determined in 2A cells by expression of cleaved caspase 3 (left panel) and annexin V binding (right panel).

Figure 5.

DNMT1 inhibition results in the suppression of STAT3 activation. (A, left) Two T-cell lymphoma cell lines (PB-1 and 2A) treated with the DNMT inhibitor DAC were examined for expression of the noninhibited DNMT1 protein (upper lane) and STAT3 phosphorylation at Tyr705 (upper middle lane) and Ser727 (lower middle lane) and for expression of total STAT3 (lower lane). Untreated cells served as a control. (A, right) Expression of noninhibited DNMT1, p-Y705STAT3, and total STAT3 in the additional T-cell lymphoma cell line, 2B, before and after treatment with DAC. (B, left) 2A cells were treated for 72 hours with 2 fluorescein-labeled DNMT1 antisense DNA oligonucleotides, designated DNMT1 AS-ON(1) and DNMT1 AS-ON(2), and non–sense DNA oligonucleotide (NS-ON) as control. Flow cytometry analysis showed efficiency of the uptake of FITC-labeled DNMT1 AS-ON(1). (B, right) Western blot expression of DNMT1, phospho-STAT3, and total STAT3 after treatment with control NS-ON, DNMT1 AS-ON(1), and AS-ON(2).